Project Summary Parathyroid hormone (PTH) is the principal hormonal determinant of the rate of bone remodeling. The long term goal of this proposal is to determine the mechanism by which PTH stimulates the production of the appropriate number of osteoblasts needed to completely refill the resorption cavities created by osteoclasts during the remodeling process. PTH increases the formation of osteoclasts by stimulating the expression of receptor-activator of NFkB-ligand (RANKL). However, the PTH-generated signals stimulating the development of osteoblasts are poorly understood. During the previous funding period, we determined in mice that infusion of either PTH or soluble RANKL (sRANKL) for 5 days increases both osteoclast and osteoblast number, but the osteoblastogenic response to sRANKL is less than that of PTH. Whereas cancellous bone volume is maintained in mice receiving PTH, reflecting balanced remodeling, infusion of sRANKL causes a decrease in cancellous bone volume, indicating that remodeling was unbalanced in favor of resorption due to inadequate numbers of osteoblasts. The greater effect of PTH on osteoblastogenesis indicates that besides the PTH-stimulated RANKL-dependent stimulus, the hormone must activate additional pro-osteoblastogenic pathways. Importantly, we have also demonstrated that PTH acts directly on osteocytes to rapidly suppress the expression of sclerostin, a potent antagonist of pro-osteogenic Wnt signaling. Gene profiling revealed that PTH, but not sRANKL, affects the expression of several components of the Wnt signaling pathway as well as pro-angiogenic cytokines. Accordingly, PTH increased marrow vascularity, but sRANKL did not. Based on these studies, and the increasingly appreciated link between the vasculature and bone formation, we hypothesize that PTH increases osteoblastogenesis via three inter-related mechanisms. One depends on the RANKL-induced increase in osteoclast number and bone resorption, which in turn increases osteoblast number via release of pro-osteogenic growth factors from the bone matrix, or via secretion of growth factors from osteoclasts themselves, or both. The second mechanism involves PTH-induced activation of pro-osteoblastogenic Wnt signaling, caused at least in part by suppression of sclerostin. Third, PTH- stimulates an increase in pro-angiogenic factors and Wnt signaling, both of which are needed for the increased development of vessels necessary for the delivery of osteoclast and osteoblast progenitors to the bone remodeling site. The experiments of Specific Objective 1 will investigate the role of Wnt signaling in the increased osteoblast number and vascularity caused by infusion of PTH. These studies will be done using DMP1-Sost mice that overexpress sclerostin, mice lacking sclerostin, and mice expressing a constitutively active mutant of the PTH receptor specifically in osteocytes. Wnt signaling will be measured by quantification of known Wnt target genes. Detailed histologic measurement of Wnt signaling will also be done in mice expressing 2- galactosidase under the control of 2-catenin (BATgal mice), as well as in BATgal mice also expressing the DMP1-Sost transgene, or lacking Sost. To facilitate future investigations, a long-term effort will be launched to develop methodology for the isolation of the pre-osteoblast targets of PTH from adult bone. Specific objective 2 will investigate the role of neovascularization in the regulation of bone remodeling. The effect of an anti- vascular endothelial growth factor antibody (Bevacizumab), or a peptide (Anginex) that blocks endothelial cell migration on bone remodeling, will be quantified under normal physiologic conditions, and following stimulation of remodeling by administration of PTH or sRANKL. PUBLIC HEALTH RELEVANCE: Project Narrative Elevated circulating levels of parathyroid hormone (PTH) are a common problem in veterans with kidney failure. Chronically elevated PTH, also called hyperparathyroidism, causes bone loss, increases the risk of fracture, and may contribute to the high mortality seen in veterans with kidney disease. Moreover, the altered mineral metabolism that characterizes hyperparathyroid patients with kidney disease may contribute to blood vessel calcification, which has also been linked with increased mortality. The proposed studies are designed to identify the PTH-regulated factors responsible for the development of the proper number of bone forming cells;and thus are vital to the VA patient care mission. Furthermore, the recognition of the PTH-regulated bone forming pathways will advance in the understanding of the mechanisms of other forms of osteoporosis such as postmenopausal and age-related bone loss.